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UseofFluo-8®AMEsters

1.LoadCellswithFluo-8®AMEsters:

AMestersarethenon-polarestersthatreadilycrosslivecellmembranes,andrapidlyhydrolyzedbycellularesterasesinsidelivecells.AMestersarewidelyusedforloadingavarietyofpolarfluorescentprobesintolivecellnon-invasively.However,cautionsmustbeexcisedwhenAMestersareusedsincetheyaresusceptibletohydrolysis,particularlyinsolution.Theyshouldbereconstitutedjustbeforeuseinhigh-quality,anhydrousdimethylsulfoxide(DMSO).DMSOstocksolutionsmaybestoreddesiccatedat–20°Candprotectedfromlight.Undertheseconditions,AMestersshouldbestableforseveralmonths.

FollowingisourrecommendedprotocolforloadingFluo-8®AMestersintolivecells.Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

a)      Preparea2to5mMstocksolutionofFluo-8®AMestersinhigh-quality,anhydrousDMSO.

b)      Onthedayoftheexperiment,eitherdissolveFluo-8®inDMSOorthawanaliquotoftheindicatorstocksolutiontoroomtemperature.Prepareaworkingsolutionof1to10µMinHanksandHepesbuffer(HHBS)orthebufferofyourchoicewith0.02%Pluronic®F-127.Formostofcelllines,Fluo-8®reagentswithaconcentrationrangingfrom4-5uMarerecommended.Theexactconcentrationoftheindicatorrequiredforcellloadingmustbedeterminedempirically.Toavoidanyartifactscausedbyoverloadingandpotentialdyetoxicity,itisrecommendedtousetheminimaldyeconcentrationthatcangeneratesufficientsignalstrength.

Note:ThenonionicdetergentPluronic®F-127issometimesusedtoincreasetheaqueoussolubilityofFluo-8®AMesters. AvarietyofPluronic®F-127solutionscanbepurchasedfromAATBioquest.

c)      Ifyourcellscontainingtheorganicanion-transports,probenecid(1–2.5mM)orsulfinpyrazone(0.1–0.25mM)maybeaddedtothecellmediumtoreduceleakageofthede-esterifiedindicators.

Note:AvarietyofReadiUse™probenecidincludingwatersolublesodiumsaltandstABIlizedsolutioncanbepurchasedfromAATBioquest.

d)      Addequalvolumeofthedyeworkingsolution(fromStepborc)intoyourcellplate.

e)      Incubatethedye-loadingplateatacellincubatororroomtemperaturefor20minutestoonehour.

Note:Decreasingtheloadingtemperaturemightreducethecompartmentalizationoftheindictor.

f)       ReplacethedyeworkingsolutionwithHHBSorbufferofyourchoice(containingananiontransporterinhibitor,suchas2.5mMprobenecid,ifapplicable)toremoveexcessprobes.

g)      RuntheexperimentsatEx/Em =490/525nm

UseofScreenQuest™Fluo-8NWCalciumAssayKitsforHTSApplications

GPCRactivationcanbedetectedbydirectmeasurementofthereceptormediatedcAMPaccumulation,orchangesinintracellularCa²⁺concentration.GPCRtargetsthatcoupleviaGqproduceanincreaseinintracellularCa²⁺thatcanbemeasuredusingacombinationofFluo-8®reagentsandafluorescencemicroplatereader.Thefluorescenceimagingplatereaders(suchas,FLIPR™,FDSSorBMGNovoStar™)haveacooledCCDcameraimagingsystemwhichcollectsthesignalfromeachwellofamicroplate(both96and384-well)simultaneously.Theseplatereaderscanreadatsub-secondintervals,whichenablesthekineticsoftheresponsetobecaptured,andhasanintegratedpipettorthatmaybeprogrammedforsuccessiveliquidadditions.BesidestheirrobustapplicationsforGPCRtargets,ourScreenQuest™Fluo-8CalciumAssayKitscanbealsousedforcharacterizingcalciumionchannelsandscreeningcalciumionchannel-targetedcompounds.

Figure2.CarbacholDoseResponsewasmeasuredinHEK-293cellswithScreenQuest™Fluo-8NWAssaykitandFluo-4NWAssayKit.HEK-293cellswereseededovernightat40,000cells/100µL/wellina96-wellblackwall/clearbottomcostarplate.Thegrowthmediumwasremoved,andthecellswereincubatedwith,respectively,100µLoftheScreenQuest™Fluo8-NWcalciumassaykitandFluo-4NWkit(accordingtothemanufacturer’sinstructions)for1houratroomtemperature.Carbachol(25µL/well)wasaddedbyNOVOstar(BMGLabTech)toachievethefinalindicatedconcentrations.TheEC₅₀ofFluo-8NWisabout1.2uM.

ComparedtoothercommercialcalciumassaykitsthateitherbasedonFluo-3orFluo-4,ourScreenQuest™CalciumAssayKitshavethefollowingadvantagesforHTSapplications:

•BroadApplications:workwithbothGPCRandcalciumchanneltargets.

•ConvenientSpectralWavelengths:maximumexcitation@~490nm;maximumemission@~514nm.

•FlexibleDyeLoading:dyeloadingatroomtemperature(ratherthan37ºCrequiredforFluo-4AM).

•NoWashRequiredandNoQuencherInterferencewithYourTargets.

•RobustPerformance:enablecalciumassaysthatareimpossiblewithFluo-4AMorFluo-3AM.

•StrongestSignalIntensity:2timesbrighterthanthatofFluo-4AM;4timesbrighterthanthatofFluo-3AM.

UseofFluo-8®Salts

Calciumcalibrationcanbecarriedoutbymeasuringthefluorescenceintensityofthesaltform(25to50µMinfluorescencemicroplatereaders)oftheindicatorsinsolutionswithpreciselyknownfreeCa²⁺concentrations.Calibrationsolutionscanbeusedbasedon30mMMOPSEGTACa²⁺buffer.Ingeneral,watercontainstraceamountofcalciumion.Itishighlyrecommendedtouse30mMMOPS+100mMKCl,pH7.2asbuffersystem.Onecansimplymakea0and39µMcalciumstocksolutionsaslistedbelow,andthese2solutionsareusedtomakeaserialsolutionofdifferentCa²⁺concentrations

A.0µMcalcium:30mMMOPS+100mMKCl,pH7.2buffer+10mMEGTA

B.39µMcalcium:30mMMOPS+100mMKCl,pH7.2buffer+10mMEGTA+10mMCaCl₂

TodetermineeitherthefreecalciumconcentrationofasolutionortheK_dofasingle-wavelengthcalciumindicator,thefollowingequationisused:

[Ca]_free=K_d[F─F_min]/F_max─F]

WhereFisthefluorescenceintensityoftheindicatorataspecificexperimentalcalciumlevel,F_ministhefluorescenceintensityintheabsenceofcalciumandF_maxisthefluorescenceintensityofthecalcium-saturatedprobe.

Thedissociationconstant(K_d)isameasureoftheaffinityoftheprobeforcalcium.Thecalcium-bindingandspectroscopicpropertiesoffluorescentindicatorsvaryquitesignificantlyincellularenvironmentscomparedtocalibrationsolutions.InsituresponsecalibrationsofintracellularindicatorstypicallyyieldK_dvaluessignificantlyhigherthaninvitrodeterminations.InsitucalibrationsareperformedbyexposingloadedcellstocontrolledCa²⁺buffersinthepresenceofionophoressuchasA-23187,4-bromoA-23187andionomycin.Alternatively,cellpermeabilizationagentssuchasdigitoninorTriton®X-100canbeusedtoexposetheindicatortothecontrolledCa²⁺levelsoftheextracellularmedium.TheK_dvaluesofFluo-8®reagentsarelistedinTable1foryourreference.

References&Citations

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